A. J. d e Koning University of Botswana, Lesotho, Swaziland Roma, Lesotho, Africa
Analysis of Egg Lipids A student project
I wish to report a series of experiments on egg yolk lipids which have been part of our laboratory course on natural products for several years. They are a n attempt to introduce to undergraduates the notoriously complex subject of lipid analysis, since most manuals and textbooks on practical organic chemistry or biochemistry do not describe any lipid experiment a t all or they do not rise above the level of a saponification-chemical or enzymatic-or an iodine value determination. The experiments are Thc isolation of egg yolk lipids and the sepnration of the phospholipid* from the non-phosphorylatedlipids. The colorinwtrw determination of the phosphorus content of both lipid fractions. The detection of choline, ethanolamine, serine, and inositol in the phospholipids by paper chromatography. The separation of the phospholipids into classes by thin layer chromatography. The determination of the fatty acid composition of the phaspholipids by gas chromatography. These experiments are the result of my own research interest and are, I believe, successful in training the students to use modem techniques for the analysis of a readily obtainable natural product. The combined experiments form a useful student project and can be completed in about eight laboratory periods of three hours duration. It is of course not necessary to complete the whole series; the thin layer and gas chromatography experiments can he conveniently omitted if the necessary equipment is not available. Isolation of Egg Yolk Lipids and the Separation of the Phospholipids from the Non-Phosphorylated Lipids Egg yolk is a rich source of phospholipids which have the general formula shown below, where XOH is choline, ethanolamine, serine, and myo-inositol.
0 0
II
R-4-0--CH
CH,-O-C-R
I I
1I 11 I
OH glycerophoapholipid
Determination of the Phosphorus Content of the Phospholipids and the Non-Phosphorylated Lipids Organic phosphorus is converted into inorganic phos-
hate bv diaestion of the livids with concentrated suliuric acib an2 hydrogen Inorganic phosphate is then determined calorimetrically by reduction of phosphomolybdic acid to molybdenum blue in a boiling water bath (2). Experimental
The lipid solutions are evaporated to dryness on a water bath. About 40 mg of the phospholipids and about 100 mg of the nonphosphorylated lipids are accurately weighed out in glass eapsules. The lipids are digested in micro Kjeldahl flasks with 1 ml and 2 ml concentrated H2S04, respectively. A few drops of HzOl are added carefully at intervals until the mixture is colorless. It is digested an extra 30 min to complete the decomposition of Hz02 and the digest transferred into 100-ml volumetric flasks which are
serine
CH@H
I
CH2-yCHah choline
myo-inositol
The non-phmphorylated lipids consist of triglycerides, cholesterol, and cholesterol esters. Phospholipids can be separated from the non-phosphorylated lipids by a variety of methods ( I ) ; a simple procedure which is adequate for our purposes makes use of the relative insolubility of the phospholipids in acetone. 48
An egg is cracked on the side of a beaker and the egg white slipped into the beaker by mefully maneuvering the yolk from one half of the egg shell to the other. The lipids are extracted from the yolk by stirring with 90 ml of the solvent mixture chloroform-methanol (21, by vol), allowed to stand for 10 miu and filtered by gravity over a Whatman No. l filter paper. The filtrate is washed once with 50 ml of a 1%aqueous NaCl solution in a separating funnel. The bottom layer consisting of a chloroform solution of the Lipids is dried over anhydrous NarS01 and filtered into a beaker. After adding a trace of antioxidant-e.g., hydmquinone or 2,6-di-tert-butyl-4-methylphenol-and a boiling chip, the solution is evaporated to near dryness on a water bath. To the residue in the beaker 30 ml of acetone is now added, and the beaker is cooled in ice for about 15 min to complete the precipitation of the phospholipids. The acetone solution of the non-phosphorylated lipids is carefully decanted into a Whatman No. 1filter paper. The semi-solid phospholipids in the beaker are washed once with 10 ml cold acetone and transferred with a spatula to a 100-ml conical flask where they are dissolved in 20 ml of petroleum ether (hp 404°C). The acetone solution of the non-phosphorylated lipids is evaporated to dryness on the water bath; the residue is also taken up in 20 ml of petroleum ether. Both flasks are labelled, a trace of antioxidant added, and stored in the refrigerator until needed.
0
CH,-O-P-OX
ethanolamine
Experimental
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Separatm of fatty ac a methyl esters tram s g yolr ~ pnospho rpds b) Chromatography on po.yelny sne g p o s.Cc#nate at 196T
gas
made up to volume. A series of 11clean and numbered test tuhes are prepared. Into tuhes 1, 2, 3, and 4, respectively, 1 ml, 2 ml, 3 ml, and 4 ml of s standard phosphate solution containing 10 rrg P/ml is pipetted. (This solution is readily prepared by dissolving 4.392 g KHzPO+in 1MX) ml water, taking an aliquot of 10 ml and making this up to 1MX)ml). One ml, 2 ml, and 3 ml is pipetted from the phospholipid hydrolysate into tuhes 5, 6, and 7, respectively, and into tuhes 8, 9, and 10, 1 ml, 2 ml, 3 ml of the nonphaspharylated lipid hydmlysate. Tube 11 serves as the blank. All the tubes are filled from a buret to a volume of 4.0 ml with distilled water and to each test tube 0.5 ml of 5 M H2S04, 0.2 ml of 5% (NH&Mo04 solution and 0.3 ml of the reducing agent is added. (The latter solution is prepared as follows: dissolve 3.75 g NanSzOl-sodium metabisulfite, 125 mg anhydrous NazSOs and acid in 25 ml water by 65 mg of 1-amino-2-naphthol-4-sulfonic warming gently over a flame. The solution is filtered and stored in the refrigerator, where it is stable for a t least a week). All the tuhes are shaken vigorously-this is important-and heated for 10 min in a boiling water bath. The tuhes are cwled and the solutions diluted to 50 ml in volumetric flasks with distilled water. The absorbance of the solutions at 630 nrn is read against the hlank with a calorimeter and a graph of absorbance versus rrg P prepared. From the graph the amount of P in tubes 5-10 is read and the percentage Pof the lipids calculated.
two main spots correspond to phosphatidylethanolamine (RF about 0.9) and phosphatidylcholine (RE about 0.4). The two minor spots correspond to sphingomyelin (Rp about 0.2) a n d lysophosphatidyl choline (Rp about 0.1). Sometimes lysophosphatidylcholine cannot be detected. Phosphatidylserine a n d phosphatidylinositol are in all probability n o t sufficiently well separated from phosphatidyl choline which appears a s a streaky spot (1, 6, 7). T h e structures of sphingomyelin a n d lysophosphatidylcholine are indicated helow.
I
C=O
I
R sphingomyelin
Detection of Choline, Elhanolamine, Serine, and Inosilol in the Phospholipids by Paper Chromatography These four water-soluble components a r e released from the phospholipids by hydrolysis with aqueous hydrochloric acid. Each substance c a n be deteeted by paper chromatography a n d is indicative of a phospholipid class (3).
Experimental About 200 mg of the phospholipid mixture is hydrolyzed by refluxing with 10 m16 M HCI for 2 hr in a 50-ml flask supplied with a reflux condenser. The mixture is filtered into a 100-ml beaker and evaporated to dryness on a water bath; the residue is taken up in 1 ml of water and filtered into a small test tube. This filtrate is used for the paper chromatographic detections. Choline. The hydrolysate (2 drops) and an authentic choline chloride solution (2 drops of a 0.5% solution in 0.1 M HC1) are spotted on the paper and an ascending ehmmatogram run overnight in the solvent system n-hutanol-acetic acid-water ( 4 1 5 , by vol, top layer). The chmmatogram is dried in air and passed through a solution of 2% phosphomolyhdic acid. The excess phosphomolyhdic acid is washed off in running tap water for 10 min, which is conveniently done by placing the paper in the sink. A solution of 0.5% SnCIa in 2 M HC1 is poured onto the paper, the paper is rinsed with running tap water to remove any remaining HC1 and dried in air. Choline shows up as a hlue spot. Its RF value is determined and compared with a literature value (4). Ethonolonine and Serine. The hydrolysate (1 dmp) and solutions of authentic ethanolamine and serine (1 drop of a 0.2% solution in 0.1 M HCI) are spotted on the paper and the ehromatogram run as described for choline. The dried paper is passed through a 0.2% solution of ninhydrin in acetone and heated in an oven at about 100°C for 5 min. Ethanolamine and serine show up as purple spots. Their RP values are determined and compared with literature data (4). In addition to ethanolamine and swine other ninhydrin reactive substances may sometimes he present; these impurities which are "soluhilized by the phospholipids can he removed, if desired, by washing the phospholipids with 0.1 M HCl(5). Inositol. The hydrolysate (about 6 drops) and a myo-inositol solution (1 drop of a 2% solution in 0.1 M HCI) are spotted on the paper and the chromatogram run as described for choline. The dried paper is passed through a mixture of 2 ml saturated aqueous AgNOa and 50 ml acetone. The paper is carefully dried a t about 60'C for 2 min and then slowly passed through a solution of 1g NaOH in 100 ml of a water-acetone mixture (1:1, by "01). The black spots are allowed to come up in air for 5 min, the paper color rinsed in runnine " water for 1 min and the hmwn hackmound . then removed with 10% aqueous NazS20a. The R , value of inasito1 is determined and compared with a literature value (4). Separation of the Phospholipids into Classes by Thin Layer Chromatography T h i n layer chromatography of the phospholipids on Silic a Gel G separates them into at least four fractions. T h e
Experimental A slurry of 25 g of Silica Gel G in 50 ml of water is prepared and poured into the spreader. Plates of 0.25 em thickness are made and then activated in the oven at 110°C for 1hr. The phospholipid mixture (1 or 2 drops of the petroleum ether solution) is spotted on the plate and developed in the solvent system chloroform-methanol-acetic acid-water (85:15:10:3, by vol). When the solvent has reached the top the plate is removed and dried in air. The plate is sprayed with a 20% ammonium hisulfate solution (prepared by dissolving 20 g (NH&SOI in 100 ml of water and adding 4 ml of concentrated HzSO*) until it is transparent. The d a t e is heated in the oven at about 220°C for 20 min. The RP values of the spurs obtained are determmed and the phospholipid compasitionesrrrnatrd from thesireof thespota.
.~
Determination of the Fatty Acid Composition of the Phospholipids by Gas Chromatography
T h e composition of the fatty acids esterified to glycerol a n d sphingosine can b e readily determined by gas chromatography. The fatty acids are first converted into methyl esters by transmethylation a n d then injected in t h e eas chromatomaph. Operating conditions for different columns a n d instruments bhviousTy cannot be given; good results are obtained in our laboratory with a Perkin Elmer F11 instrument using polyethylene glycol succinate as stationary phase at 1 9 6 T a n d helium as carrier gas. A typical example of t h e separation achieved is shown in the figure. Experimental The phasphoiipid mixture (100-200 mg) is weighed out in a 50ml flask and refluxed for 90 min with the methylating mixture (methanol-benzene-sulfuric acid 20:10:1, by vol. A boiling chip and a few crystals of antioxidant are added ta the solution. The reaction mixture is transferred to a separating funnel with 50 ml of petroleum ether (bp 40-60°C) and washed three times with 20 ml of water. The water layers are discarded and the top layer is dried over anhydrous NazSO1, filtered, and the petroleum ether evaporated on the water bath. The residual methyl esters are taken up in about 1 ml of petroleum ether and kept for gas chmmatography. The metKyl esters are run on tho gas chromatograph and the acids identified by means of their relative retention times, which should be compared to literature values (a), (see also the figure). Volume 51. Number 1, January 1974
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49
A graph of retention time versus the logarithm of the carhon number is plotted. Note that this straight line relationship can in some instances he used for identification of acids. The chromatogram is traced on tracing paper, the peaks cut out and weighed. The fatty acid composition is calculated and compared with s literaturevalue (9), (see also the figure).
Literature Cited (1) Ansell. G. B., and Haathome, J. N., "Phepholipida." Ekvivivi Publ. Comp., Amstcrdrrm, 1964. p. 10. (2) Bmtiett, G. R.,J Bid Chrm., 234,466 (1959).
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Journal of Chemical Education
(3) deKming. A. J . , J Sci. FoodA#r., 17,112 (1986). (4) Ledem, E.. and Ledenr, M., "Chmmatcma~h~,"E h 6 . r ~ v b cornp., i ~ ~ ~ dam, 1957. (5) deKoning,A, J.,Bbc~im,BbPhYs.Aefo,84,467,19M), (6) c w r , M. I.. and Jam*, A. T., '.tipid ~ i ~ ~ h s ~ ian~ 1otm6~cti0~; try: chapmsn and Hall Ltd.. London. 1371. (7) M e n e t t i , G. V., "Lipid Chmmatasrapbic Analysis," Vol. 1, Marcel Dokkcr. I~c., NouYork. IS67. (8) h u h a r , J. W , I ~ U I IJ ~ w.. , h n , P., stoffel, w., and i\hrrask,E. H.,N U ~ C Reus., Suppl.. 11, VA(1959). (9) Gunstane, F. D.. "An lntmdvction to the Chemistry and BbeEhsmistn, of Fatty Acids and thci Glynridea," h d Ed.. Chspmm and Hall Ltd., London. 1967, p. 173.
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